Infertility constitutes a global medical problem, with male infertility contributing to half of all cases. Moreover, more than 70% of male infertility cass are considered idiopathic. This huge gap in our understanding of etiology of male infertility is partially attributed to our insufficient knowledge of physiology of spermatozoa, and human sperm cells in particular. While delicate regulation of sperm physiology occurs on many levels, its regulation by bioactive lipids deserves special attention. Our preliminary data indicate that several bioactive lipids influence the activity of sperm ion channels, which in turn regulate sperm motility and the acrosome reaction. For example, the steroid hormone progesterone stimulate the opening of the primary calcium channel of human sperm, CatSper, and controls sperm intracellular Ca2+. Progesterone also inhibits potassium sperm channel KSper. Our preliminary data indicate that CatSper channel can be inhibited by certain fatty acids, but also in addition to progesterone, can be up-regulated by an unknown sperm endogenous lipid easily extracted by fatty acid free bovine serum albumin (faf BSA) and -cyclodextrin. A brief incubation of sperm with either faf BSA or -cyclodextrin results in inhibition of CatSper activity. These experiments were done under conditions when sperm cytoplasm is removed, but sperm membrane stays intact. Moreover, such action is reversible indicating that the molecule responsible for activation of CatSper can be synthesized by intact sperm plasma membrane in the absence of secondary messengers. In addition, our recently published data indicate that many sperm ion channels and the molecules that modulate their activity are specific to human spermatozoa, and either are not observed, or their functions are altered in murine spermatozoa. This highlights the necessity to concentrate efforts of this proposal on studying bioactive lipid signaling in human spermatozoa specifically. In order to identify the molecule(s) responsible for sensitizing CatSper channel we will perform lipid extraction from human sperm plasma membrane by -cyclodextrin in order to deplete them from CatSper sensitizer, wait for the later to recover, and again extract lipids with -cyclodextrin. The newly synthesized lipids will be identified and tested for their ability to modulate CatSper. We will also obtain bioactive profiling of human ejaculated and capacitated spermatozoa. The knowledge gained from the proposed research will fill in gaps in our understanding of the basic mechanisms underlying the regulation of human sperm by bioactive lipids, may lead to the development of novel diagnostic tests for male infertility, and to the development of male contraceptives.